transcript o mics

8/22/2019 Transcript o Mics

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Transcriptome

analysis


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Genomics Transcriptomics Proteomics

Bioinformatics


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The set of all RNA molecules, including

mRNA, rRNA, tRNA, and other non-coding

RNA produced in one or a population of cells.

The transcriptome can vary in different tissues andwith external environmental conditions.

Transcriptome


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Transcription

The process of creating an equivalentRNA copy of a sequence of DNA.


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mRNA processing


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Reverse transcriptase, also known as RNA-dependent DNA

polymerase, is a DNA polymerase enzyme that transcribes

single-stranded RNA into double-stranded DNA.


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cDNA synthesis for 5 RACE

(Rapid Amplification of cDNA Ends)


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Northern blotting


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Accumulation of CBFtranscripts in response to low temperatures.


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Fig. RNA gel blot analysis of stress-inducible genes. Each lane was loaded with 10 g of total RNA isolated from two-week-old rice

seedlings that were each exposed to H2O, dehydration, 250 mM NaCl, 100 M ABA and 4? cold treatment for 1, 2, 5, 10 and 24

hours. RNA was analyzed by gel-blot hybridization using gene-specific probes based on selected stress-inducible clones obtained via

rice cDNA microarray analysis. Stress-inducible clones were classified into various groups on the basis of their expression patterns in

RNA gel blot analysis under each stress treatment. Some of the inducible genes were induced by all four stress-treatments while some

were upregulated by cold, drought and high salinity, and others were induced by drought and high salinity. Some genes were induced

by singular conditions such as cold only


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Dot blot analysis


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cDNA library normalization by Trimmer Kit


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Cold treatment

Subtractive cDNA library


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A B

C

D

1 2 3 4 5 6

M

MM

M


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Cold up-regulated library

Cold down-regulated library


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Gene Ontology Statistics of Cold stressed EST libraries

Unknown9.9%

Transport

4.8%

Transcription

1.2%

Metabolic

processes

18.1%

Signal

transduction

0.5%

Protein

metabolism

15.5%

Cellular

processes

10.4%

Development

0.7% Energy

pathways

5.1%

Cell

organization

1.0%No hit

23.4%

Stress

9.4%

DNA binding

11% Hydrolase

6% Kinase

2%

Other binding

10%

Transporters

7% Transferase

4%

Stuctural

molecules

9%

Protein binding

5%

Other molecular

function

1%

Enzyme activity

16%Unknown

function

10%

No hit

19%

Unknown

7%

Ribosome

3%Membrane

11%

Cytosol

8%

Mitochondria

7%

Nucleus

2%

Other cellular

components

5%

Plastids

31%

No hit

25%

Cell wall

1%

Mitochondria

4%

Other cellular

components

5%

No hit

47%Cytosol

4%

Plastids

18%

Cell wall

1%

Membrane

9%Ribosome

2%

Unknown

8%

Nucleus

2%

Unknown

7%

No hit

46%

Stress

7%

Metabolic

processes

13%

Transcription

1%

Transport

3%

Cell organization

2%

Development

1%

Protein

metabolism

10%

Energy pathways

2%Cellular processe s

7%

Signal

transduction

1%

Unknown

9% Protein binding

3%

Other molecular

function

1%

Enzyme activity11%

Other binding

6%

Stuctural molecules

6%

Transferase

4%Transporters

3%

No hit

43%

Kinase

2%

Hydrolase

5%

DNA binding

7%

Up-regulated library Down-regulated library

Biological

processes

Molecularfunctions

Cellular

components


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EST and Unigene sets in Crop plants


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Microarray Analysis

DNA sequences arranged in a matrix

Tests for binding to complementary

sequences


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Why DNA Microarray technology?

DNA microarray gives snapshot of mRNA

expression in a genome at a particular time

Can take multiple snapshots to watch changing

patterns of mRNAs over time in different

developmental stages, tissues, environmental

stresses

Identifying new targets for functional genomics


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Steps in Microarray Analysis

Arrange DNA or cDNA in an ordered array on aglass slide

(can also build oligonucleotides directly on a slide)

Incubate array with fluorescently-labeled probe

Detect probe hybridization using a laser

Record and store data with computers


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cDNA arrays Oligonucleotide arrays

Gene expression analysis using DNA microarrays

Oligo

array

Spotted

array


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cDNA Microarray


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Transforming images into data

Test sample labeled red(Cy5)

Reference sample labeled

green (Cy3)

Red :gene overexpressedin

test sample

Green :gene underexpressed

in test sample

Yellow- equally expressed

red/green- ratio of expression


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Affymetrix

Wafer and Chip Format

1.28cm

5 - 50 m

5 - 50 m

Millions of identicaloligonucleotide

probes per feature

49 - 400

chips/wafer

up to ~ 3,000,000 features/chip


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Procedures for Target Preparation

cDNA

Wash & Stain

Scan

Hybridise

(16 hours)

RNAAAAA

B B B B

Biotin-labeled

transcripts Fragment

(heat, Mg2+)

Fragmented cRNA

B B

B

B

IVT(Biotin-UTP

Biotin-CTP)


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GeneChipExpression Analysis

Hybridization and Staining

Array

cRNA Target

Hybridized Array

DetectionStreptavidin phycoerythrin


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Differential expression patterns

15 4 3 2 1.5 1:1 1.5 2 3 4 15

Foldchange

Phl A B C D E

-1

-2

12

3

0

4

-1

-2

1

2

3

0

4

-1

-2

1

2

3

0

4

-1

-2

1

2

3

0

4

-1

-2

1

2

3

0

4

Phl A B C D E

I

II

III

IV

VI

VII

IX

VIII

V X


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A B

Cotton plants grown in the fields

under rainout shelter. (A) Control plot

and (B) Drought stress induced plot.

Different stages of cotton samples

selected for transcriptome

analysis.

Leaf 0 dpa

5 dpa 10 dpa 20 dpa

Transcriptome analysis of cotton (G. hi rsutum)

during boll development under drought stress


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Cluster analysis of differentially expressed genes

in different stages

In total 4,522genes accounted for

19%of the genes on the cotton GeneChip were differentially expressed.

Out of 4,522 differentially

expressed genes 2,491genes were up

regulated and 2,419genes were down

regulated.

Gene expression pattern among the three boll


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Gene expression pattern among the three boll

developmental stages under drought stress

10 dpa

20 dpa

5 dpa


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01 METABOLISM

14%

02 ENERGY

1%10 CELL CYCLE AND DNA

PROCESSING

3%

11 TRANSCRIPTION6%

14 PROTEIN FATE (folding,

modification, destination)

3%

16 PROTEIN WITH BINDING

FUNCTION OR COFACTOR

REQUIREMENT (structural orcatalytic)

11%

18 REGULATION OF METABOLISM

AND PROTEIN FUNCTION

1%

20 CELLULAR TRANSPORT,

TRANSPORT FACILITIES AND

TRANSPORT ROUTES

3%30 CELLULAR

COMMUNICATION/SIGNAL

TRANSDUCTION MECHANISM

3%

32 CELL RESCUE, DEFENSE AND

VIRULENCE

7%

34 INTERACTION WITH THE

ENVIRONMENT

8%

36 SYSTEMIC INTERACTION WITH

THE ENVIRONMENT

7%

40 CELL FATE3%

41 DEVELOPMENT (Systemic)

1%

42 BIOGENESIS OF CELLULAR

COMPONENTS

4%

70 SUBCELLULAR LOCALIZATION

19%

99 UNCLASSIFIED PROTEINS

7%

Functional categories of common differentially expressed

genes in different boll development stages under drought

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0

5

10

15

20

25

leaf

(-5)dpa

5dpa

10dpa

20dpa

Functional categories of differentially expressed

genes in different stages of drought induced samples

Functional categories

Percentageofgene

s

(http://mips.gsf.de/projects/funcat)


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